The major goal of this research project is to understand the molecular mechanism of tumor suppression by cytoskeletal proteins, and in particular, Tropomyosin (TM). Earlier work from this laboratory established that suppression of TM levels are closely associated with neoplastic transformation. More recent work has demonstrated that restoration of expression of one of the two isoforms, TM1, was adequate to revert the malignant phenotype of v-Ki-ras transformed murine fibroblasts (DT cells). These studies were extended to examine the role of another isoform of TM, TM2, in suppressing the ras transformed phenotype of murine fibroblasts. A cDNA encoding TM2 was isolated from NIH3T3 cells by RT-PCR method and subcloned into an eukaryotic expression plasmid. Transfectants of DT cells which expressed TM2 protein (DT-TM2) resembled morphologically DT cells and grew efficiently in anchorage independent fashion, indicating that TM2 protein does not posses tumor suppressive property. In light of the reports that the 3' untranslated sequences of TM gene, but not the proteins, function as tumor suppressor elements, we have reevaluated the role of TM1, a product of TM gene, as a tumor suppressor. DT cells, transduced with retrovirions carrying protein coding sequences of TM1 (TM) expressed elevated levels of TM1 protein, displayed flat morphology and failed to manifest anchorage independent growth. Tumor growth in athymic nude mice was possible only in the absence of expression of TM mRNA, unlike the case with TM2. Thus, the two major isoforms of TM differ in their ability to revert ras transformed NIH 3T3 cells. TM1 is considered to be a putative tumor suppressor gene. Our previous studies showed that suppression of TM1 is a common feature in neoplastic transformation by oncogenes. To test if TM1 functions as a general anti-oncogene, effect of elevated TM1 levels are being examined in another oncogene-transformed fibroblast cell line. Cell lines of v-src oncogene transformed-NIH3T3 cells that over express TM1 have been generated and are being characterized.